Medicinal association of a biguanine and a carrier, for example metformin and arginine

ABSTRACT

The present invention relates to a compound of formula III, 
                         
method of making the same and its use in the treatment of diabetes.

FIELD OF THE INVENTION

The present invention relates to a medicinal combination of two activeprinciples, having, jointly, a complementary and/or synergistic action,this being for the treatment of diabetes, in particular of type 2diabetes.

The expression “complementary action” is intended to mean thepharmacological action of two different compounds making it possible toact on the same pathology via two respectively different pharmacologicalmechanisms, for example the combined use of two anti diabetic agents,such as a biguanide and a sulfonylurea.

The expression “synergistic actions” is intended to mean thepharmacological action of two compounds, consisting in potentiating theaction of at least one of said compounds, for example potentiation ofthe action of a biguanide by the action of a transporter as describedand proposed hereinafter in the invention.

BACKGROUND OF THE INVENTION

It is known that metformin in the form of hydrochloride is the firstchoice medicine in the treatment of hyperglycemia and of non-insulindependent diabetes. This metformin hydrochloride is used alone or incombination with a sulfonylurea, an alpha-amylase inhibitor or aglitazone.

Metformin hydrochloride at the dose of 50 mg/kg in rats is active onconventional models of non-insulin dependent diabetes such as thestreptozotocine model and the fructose model.

It has low bioavailability (60%) and its entry through the intestineoccurs preferentially in the jejunum and in the ileum. This lowbioavailability explains the bothersome side effect of metformin, namelydiarrhea.

Metformin is a very basic biguanide which is completely ionized atintestinal pH values. Its entry therefore involves a physiologicaltransporter system, which explains the preferential entry.

SUMMARY OF THE INVENTION

An object of the present invention is to reduce the side effects and toimprove the bioavailability of the metformin by acting on thisphysiological transporter system.

Surprisingly, the present invention has demonstrated that, whenassociated with metformin, an amine or polyamine, having structuralsimilarity with a biguanide, and, in particular with anN-dimethylbiguanide, has indeed this role of transporter with respect tothe metformin. More particularly, among transporters, arginine ispreferred because of the similarity of its chemical structure withmetformin. Specifically, L-arginine (see FIG. 3) activates itself itsown intestinal transfer. In addition, arginine is the precursor for thesynthesis of the nitroso radical NO, which is recognized as being one ofthe most potent vasodilators, both of veins and of arteries, and for itshemodynamic and hemorheological properties. This action characteristicof arginine might be beneficial for pathologies secondary to thedevelopment of diabetes, namely macroangiopathies, microangiopathies,neuropathies, nephropathies and retinitis of diabetics.

In accordance with the document “Marfella R. et al., Metformin improveshemodynamic and Theological responses to L-arginine in NIDDM patients,Diabetes care, 1996 sep 19(9) 934-9”, an experiment is described inhumans, outside any clinical or therapeutic protocol, consisting inadministering by the intravenous route (under infusion) a total dose of30 g of arginine over 30 min, which is enormous and unachievable incommon therapy.

In accordance with this same document, it has been shown that thesimultaneous administration of metformin potentiates the hemodynamic andhemorheological action of aginine. However, by contrast, the therapeuticeffects specific to metformin, in particular the antihyperglycemiceffects, are not modified by arginine.

Thus, the results obtained according to the invention, on experimentalmodels of diabetes, have shown that metformin active compounds having,within their structure, an arginine residue, were capable of increasingthe bioavailability of the metformin, in an unexpected manner, bypotentiating its effects against hyperglycemia, which is the mainsymptom of diabetes.

All the biguanides currently used, or being developed, in the treatmentof diabetes have the side effects and the problems of bioavailabilitycited above.

Among the amines or polyamines which have a structural similarity withbiguanides, and which can be combined as a transporter with thesebiguanides, those which are natural for the human physiology, i.e.biogenic, and/or those which are pharmacologically and therapeuticallyactive, are preferred.

Among the pharmacologically and therapeutically active amines orpolyamines, those belonging to the same therapeutic class as metformin,or those making it possible to act on a pathology which is associatedwith diabetes are preferred.

Among the amines or polyamines which are natural for human physiology,i.e. biogenic, those which are metabolizable, biodegradable byintegration into an endogenous metabolic cycle, into conventionalmetabolites will be preferred.

The present invention relates to a medicinal set for the treatment ofdiabetes, in particular of type 2 diabetes, combining a biguanide, inparticular an N-dimethylbiguanide, as a first medicament, and an agentfor transporting the said biguanide, as a second medicament, said setcomprising:

-   -   a) a therapeutically active quantity of the biguanide    -   b) and a quantity of transporting agent, previously determined        to potentiate the therapeutic activity of the biguanide        according to (a).

It also relates to a set as described above, characterized in that thebiguanide according to (a) and the transporting agent according to (b)are in an equimolar quantity.

It also relates to a set as described above, characterized in that thebiguanide is metformin.

It also relates to a set as defined above, characterized in that thetransporting agent is a biogenic amine, for example chosen from thegroup consisting of arginine, putrescine, cadaverine, spermidine,spermine, and is for example L-arginine.

It also relates to a set as described above, characterized in that thebiguanide and/or the transporting agent are in a form suitable for theircontrolled release respectively,

The set according to the invention, chemically put together for thecontrolled release of at least biguanide, is also characterized in thatit is an active compound of general formula A′

V′

C′, capable of restoring at least the entity A by cleavage, in vivo, ofthe corresponding attachment between A′ and V′, it being specified that:

-   -   V is a biogenic vectorization compound, of general formula        X—R—Y, in which,    -   R represents an aliphatic, cyclic or alicyclic, saturated or        unsaturated hydrocarbon chain of 2 to 10 carbon atoms, which is        optionally substituted with C1 to C5 alkyl groups and/or        hydroxyl groups,    -   X and Y are each a free acid, amine or alcohol function.

A and C are two respectively different active principles, namely thebiguanide and the transporter, one of which comprises a chemicalfunction complementary to the function X, capable of reacting with thelatter so as to give an ionic A′ - - - V′ or covalent A′-V′ attachmentwhich can be cleaved in vivo, and the other of which comprises achemical function complementary to the function Y, capable of reactingwith the latter so as to give an ionic V′ - - - C′ or covalent V′-C′attachment which can be cleaved in vivo.

The set defined above is in a variant according to the invention,characterized in that, the V′ - - - C′ or V′-C′ attachment can becleaved in vivo, and said active compound is also capable of restoringthe entities V and C by said cleavage in vivo.

The invention also relates to an active compound as defined above, Abeing metformin, characterized in that the metformin is attached to thebiogenic vectorization compound by salification of the terminal primaine[sic] amine function of the metformin.

The invention also relates to an active compound as defined above,characterized in that when C is arginine, the arginine is attached tothe biogenic vectorization compound V using an acylation reaction.

The invention also relates to an active compound as defined above,characterized in that V is chosen from among the set of diacidsconsisting of oxalic, malonic, succinic, glutaric, adipic, pimelic,suberic, azelaic, sebactic [sic], malic, isatic and phthalic acids, andpreferably succinic acid.

The invention also relates to an active compound of formula III

The invention also relates to the use, as medicine, of a compound asdefined above.

It relates, more particularly, to the use, as medicine, of a compound ofgeneral formula A′

V′

C′ as defined above.

The invention also relates to a process for preparing an active compoundaccording to the invention, comprising the following steps:

a) reaction for condensation and/or salification of the biogenicvectorization compound V with one of the active principles A or C,

b) reaction for condensation and/or salification of the product of thereaction according to (a) with the other active principle C or A.

Another subject of the invention is a pharmaceutical compositioncomprising at least one compound according to the invention, incombination with one or more compatible and pharmaceutically acceptablevehicles, diluents, excipients or adjuvants.

Another subject of the invention is also the pharmaceutical compositionas defined above, which makes it possible to adjust a daily dose inhumans of between approximately 0.2 g and approximately 1 g of the saidactive compound, to one or more doses.

In the antidiabetic or antihyperglycemic indication, a medicinal set (ortherapeutic treatments kit) according to the invention, and inparticular an active compound of formula A′

V′

C′ as defined above, may be combined with another antihyperglycemicagent such as a sulfonylurea.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-3 show exemplary formulae for the arginine, the metformin andthe salt of the arginine hemisuccinimide with the metformin,respectively.

DETAILED DESCRIPTION OF THE INVENTION

The expression “biguanide” is understood to mean in particularN-dimethylbiguanides, substituted or otherwise, and for examplemetformin, but also other pharmaceutical compounds, for example buforminor fenformin.

Preferably, the biguanide is metformin.

The expression “simultaneous administration” is intended to mean theadministration, in a single dose, of the two active principles, it beingunderstood that the simultaneous administration allows the release, inthe organism, of the two active principles simultaneously or insequence.

The term “biogenic” is intended to mean a chemical compound which is ofnatural or unnatural origin and/or is metabolizable and/or isbiodegradable and/or is atoxic with respect to the human or the animal,at a physiological dose.

The term “transporter” is intended to mean a molecule or substance whichallows the transfer of another molecule across a barrier, either byforming an attachment, or without forming an attachment, by activatingthe transport system, for example by protein induction, activation ofoxygen-dependent ATPase systems or by substance exchange.

More particularly, the term “transporter” is intended to mean herein anymolecule or substance making it possible to potentiate the entry of abiguanide such as metformin, and therefore to facilitate the transportthereof in the jejunum.

For the implementation of a medicinal combination as defined above,various administration solutions can be considered, such as for example:

-   -   a pharmaceutical formulation or presentation making it possible        to administer, in a single dose, both the biguanide and the        transporter    -   or two respectively different pharmaceutical presentations        making it possible, in a suitable packaging, to administer, at        the same time and respectively, both a dose of biguanide and a        dose of transporter.

Quite particularly, and preferably, this implementation is carried outusing an active compound of general formula A′

V′

C′, capable of restoring the entities A, V and C by cleavage, in vivo,of the corresponding attachments between A′, V′ and C′, it beingspecified that:

-   -   V is a biogenic vectorization compound, of general formula        X—R—Y, in which,    -   R represents an aliphatic, cyclic or alicyclic, saturated or        unsaturated hydrocarbon chain of 2 to 10 carbon atoms, which is        optionally substituted with C1 to C5 alkyl groups and/or        hydroxyl groups,    -   X and Y are each a free acid, amine or alcohol function.

A and C are two respectively different active principles, namely thebiguanide and the transporter, one of which comprises a chemicalfunction complementary to the function X, capable of reacting with thelatter so as to give an ionic A′ - - - V′ or covalent A′-V′ attachmentwhich can be cleaved in vivo, and the other of which comprises achemical function complementary to the function Y, capable of reactingwith the latter so as to give an ionic V′ - - - C′ or covalent V′-C′attachment which can be cleaved in vivo.

The expression “cleavage in vivo” is intended to mean herein all formsof chemical hydrolysis, likely to be observed in vivo, for example, acidhydrolysis and enzymatic hydrolyses by amidases or esterases, forexample.

The expression “complementary chemical function” is intended to mean anychemical function capable of reacting with a free or terminal functionof the biogenic compound. For example, V has to comprise a functionwhich reacts with A (biguanide) and a function which reacts with C(transporter). Thus, if A and C each have an acid function, V is adiamine, a dialcohol or an alcohol-amine, so as to form, respectively,an amide, an ester or a salt. Thus, if A and C each have an aminefunction, V is a diacid so as to form an amide or a salt. If A and Ceach have an alcohol function, V is a diacid so as to form a diester.With this principle, all compositions are possible. Consequently, if Ahas an acid function and C an alcohol function, V is, for example, analcohol-amine, so as to act with the acid function of A to give anamide, an ester or a salt, and with the alcohol function of C to give anester.

The expression “covalent attachments” is herein intended to meanchemical attachments capable of being formed, by reaction of so-calledcomplementary chemical functions, between the biogenic vectorizationcompound V and the active principles A (biguanide) And C (transporter).

The expression “ionic attachments” is herein intended to meanattachments via electrostatic force, capable of being formed, by actionof the so-called complementary chemical functions, between the biogenicvectorization compound V and the active principles A (biguanide) and C(transporter), therefore attachments of the acid salt, amine salt,alkoxide and acid/base type, and this being independently of the molarproportion existing between the compound V and the active principle A orC, belonging to the complex formed by said ionic attachments.

The expression “attachment which can be cleaved in vivo” is intended tomean any attachment which allows the release or restoration of theactive principles A (biguanide) and C (transporter), and of the biogenicvectorization compound V, in vivo, by breaking the ionic or covalentattachments between the complementary chemical functions of A and V, andof C and V.

The covalent attachments which can be cleaved are cleaved by the actionof the enzymes present in the in vivo medium of the site of release.Since these covalent attachments are amide attachments or esterattachments, the enzymes involved in this cleavage are amidases,esterases and hydrolases. These enzymes are present in particular in thedigestive tract (oral administration), predominantly in the liver and inthe bloods and are potentially present in the target organs.

Amidases which hydrolyse the attachment —CO—NH— are found in the liver,they are relatively inactive; hence an expected sustained effect withthe compound according to the invention bearing such an attachment.Among these amidases, some are known; they are endopeptidases whichhydrolyse gamma-amine-containing or gamma acid attachments. According tothe invention, V can in fact be a gamma-amino acid, with a second acidor amine function in the gamma position (in the case of glutamic acid orof lysine, for example).

Esterases which hydrolyse the attachment —CO—O— are extremely numerousin living organisms. They are, however, ubiquitous and relativelynon-specific for a substrate, hence a high reaction rate, with rapidrelease of the constituents A (biguanide), V, C (transporter) of theactive compound according to the present invention. Those most specificfor a substrate bear the name of this substrate and, by way of this,mention may be made, for example, of cholinesterases or procaineesterases.

Hydrolases also hydrolyse esters and all large molecules supplied to theorganism in the form of foods. These hydrolases are numerous andubiquitous also. They will, however, be specific for the biogenicvectorization compound V used.

As cleavage enzymes which can be used for implementing the presentinvention, mention may be made of proteolytic enzymes such as pepsin,trypsin, catalases, and endo- and exopeptidases. Enzymes which can alsobe used are amylases and osidases, and finally lipases andbeta-oxygenases for the destruction of lipids.

These enzymes are involved only when the structure of the biogenicvectorization compound comprises one or more attachments which they arecapable of cleaving. For example, the lipase acts if the biogenicvectorization compound is a long chain diacid (8 to 10 carbon atoms,comparing it to a fatty acid), and the A-V or V-C attachment is obtainedby condensation with a secondary alcohol function of A or of C.

The ionic attachments which can be cleaved are cleaved as a function oftheir site of release, for example intestine, liver, plasma or targetorgan, it being understood that acid salts or amine salts or alkoxidesare generally ionized at the pH is of the media of living organisms.Generally, the pH is between 2 and 8 and is, for example, 2 for thestomach and 6, for example, for the intestine.

There is therefore ionization of the active compound according to theinvention, as a function of the type of salt used, and a dissociation ofsaid active compound, when the latter comprises at least one ionicattachment. The salt is chosen as a function of its dissociationconstant and of the pH of the in vivo site of release. For example, fordissociation in the stomach, a salt of a weak acid and of a strong baseis chosen.

The choice of the biogenic vectorization compound, and in particular thechoice of its free functions X and Y, is made according to the nature ofthe free and complementary chemical functions present in or on theactive principles (biguanide and transporter) intended to be vectorized,i.e. attached by covalent or ionic attachment to this biogenicvectorization compound, but also according to the sites of cleavage andrelease chosen. This biogenic vectorization compound is a product whichis of natural or unnatural origin and/or is metabolizable and/or isbiodegradable and/or is atoxic with respect to the human or to theanimal, at a physiological dose. This biogenic vectorization compoundwill be chosen from biologically tested and described compounds, such asgamma-amino acids involved in protein synthesis, biacids involved in theKreps [sic] cycle and ethanolamines constituting cell membranes, whichare metabolizable and atoxic, and capable of being integrat d,themselves or their metabolites, into the major biological cycles oflife. By way of a biogenic vectorization compound, mention may be made,for example, of succinic acid which is found in the Kreps [sic] cycle ormethyl succinic acid which is biodegraded to succinic acid.

The attachments selected, between the biogenic vectorization compoundand the combined active principles according to the present invention,that is to say, biguanide and transporter, depend on the possiblemetabolisms at the gastrointestinal and hepatic level.

For example, the salts can be dissociated in the digestive tract, thehydrolysis possibly being delayed using gastro-resistant pharmaceuticalforms. The esters are hydrolysed in acid medium, or hydrolysed by theesterases of the gastric juices, the hydrolysis also possibly beingdelayed using gastro-resistant pharmaceutical forms. The amides arehydrolysed by the hepatic amidases, the kinetics of these hydrolysesbeing generally slow.

Various assays can be carried out in order to evaluate the ability ofthe A′

V′ and V′

C′ attachments to be cleaved in vivo and of the active principles A(biguanide) and C (transporter) to be correspondingly released. Theseassays consist, for example, in observing the release of the activeprinciples in an intestinal juice, or studying the hepatic metabolism onrat hepatocyte primary cultures. These two tests are described below.

In vitro Assay of Cleavage in an Intestinal Juice

A preparation of intestinal juice containing trypsin, peptidases,lipase, amylase and all the other enzymes of the exocrine pancreas isused. This assay is validated beforehand using calibration compounds. Aknown amount (of the order of one microgram) of the compound A′V′C′ ismixed together with a known amount of intestinal juice (the trypsin andlipase contents of which are controlled). The reaction mixture is keptat 37° C. for one hour. This time is compatible with the intestinaltransit. Samples are taken every 15 min, and the products A and C aredetected and their concentration measured using HPLC coupled to a UVdetector, or a mass spectrometer if it is not possible to use UV. Thecolumns used depend on the nature of A and of C, but are generallyion-exchange columns, because of the presence of released alcohol, amineor acid forms. After calibration, the total amount of A (biguanide) orof C (transporter) released in 1 hour is determined, and theintermediate points make it possible to calculate the dissociationconstants Km and the rate Vmax of the enzymes for the active compoundA′V′C′ used. This assay can be coupled with determination of the releaseof A (biguanide), C (transporter) and V in the gastric juice, usingexactly the same principle but replacing the intestinal juice withgastric juice.

In vitro Assay on Rat Hepatocyte Primary Cultures

A primary culture of rat hepatocytes, which are close to those of humansfor metabolism studies, is used, in a HEPES medium to which a knownamount of compound A′V′C′, of the order of one microgram, is added. Theproducts are left in contact for 6 hours, and samples are taken, at 1hour, 2 hours and 4 hours, on which the supernatant is isolated and thehepatocytes in the pellet are lysed. In these media, the concentrationsof active principles A (biguanide) and C (transporter) released aremeasured. As previously, it is possible to calculate the Vmax and Km ofthe enzymes involved in the metabolism.

When the compounds according to the invention do not cross the cellmembranes, the same type of study can be carried out on a rat liverhomogenate.

The possible toxicity of the biogenic vectorization compound is relatedto that of the active compound (A′V′C′) according to the invention. Asthis active compound is metabolized to A (biguanide), C (transporter)and V, and V is a substance which is by definition biological, thetoxicity of the compound according to the invention must compare to thesum of the toxicities due to the administration of the biguanide A andof the transporter C. In addition, when the active compound combines twoactive principles having, under these conditions, at least for oneactive principle, an efficacy greater than that of said same activeprinciple alone, said compound can be considered to be less toxic.However, a method for predicting the toxicity, alternative to thestandard in vivo methods, is proposed hereinafter for comparing thetoxicity of A and of C and of A′

V′

C′ at identical concentrations expressed as A or as C (see ToxicologicEmergencies, Sixth Edition 1997, Goldfranck et al. Appleton and Lange,Conn., USA).

In Vitro Toxicity Assay

A method for culturing primary hepatocytes over a 96-hour period is used(see Biochemical Pharmacology, Vol. 50, 1995, pp 775-780). Thehepatocytes are isolated in situ by collagenase perfusion. They are thenplaced in a Williams medium supplemented with foetal calf serum, withcortisol and with glutamine, in a proportion of 1 million cells perwell. Increasing and toxic concentrations of A (biguanide) and C(transporter) and of A′

V′

C′ are added to each well. Samples are taken after 6 h, 12 h, 24 h, 48 hand 96 h, and, the viability of the cells is determined with a methyleneblue test, by albumin expression, by hepatocyte apoptosis and bymeasuring cytochrome P450 activity.

The viability of the cells with the methylene blue test gives resultssimilar to those obtained with an LD₅₀.

The results obtained with the albumin expression make it possible tolearn the limits of tolerance of the hepatocyte to any toxic substance(end toxicity). Specifically, one of the main roles of the hepatocyte isto synthesize proteins. During a toxic effect, this expression ofalbumin synthesis and release is modified.

The results obtained with hepatocyte apoptosis make it possible toconfirm the end toxicity, since during a contact with a toxic substance,the cells will program their destruction, which corresponds to thephenomenon of apoptosis which is measured by the abnormal DNA.

The measurement of cytochrome P450 activity documents the phenomena ofinduction and of inhibition of these enzymes, often encountered withpharmacologically active products. A series of assays makes it possibleto determine the activity of the cytochrome P450 isoforms.

The present invention also relates to the following variants:

-   -   A is metformin in the abovementioned general formula, the        metformin is attached to the biogenic vectorization V compound        by salification of the terminal primary amine function of the        metformin    -   C is arginine in the abovementioned general formula, the        arginine is attached to the biogenic vectorization compound V        using an acylation reaction    -   V, in the abovementioned general formula, is chosen from the set        of diacids consisting of oxalic, malonic, succinic, glutaric,        adipic, pimelic, suberic, azelaic, sebactic [sic], malic, isatic        and phthalic acids, and preferably succinic acid

The invention also relates, as medicine, to the compound of formula III.

The invention also relates to any pharmaceutical composition comprisingan active compound as defined above, in combination with one or morecompatible and pharmaceutically acceptable vehicles, diluents,excipients, or adjuvants. Preferably, such a pharmaceutical compositionmakes it possible to adjust a daily dose in humans of between 0.2 g and1 g of each active principle (biguanide or transporter), to one or moredoses. For example, gastro-resistant pharmaceutical forms can be used inorder to avoid any hydrolysis in the stomach.

Preferably, an active compound as defined above can be obtained at theend of the following steps;

-   -   reaction for condensation and/or salification of the biogenic        vectorization compound (V) with one (A or C) of the active        principles,    -   reaction for condensation and/or salification of said condensed        and/or salified biogenic compound obtained with the other active        principle (C or A).

Conventionally, the condensation reactions which can be used are amineacylation reactions and alcohol esterification reactions.

When one (A or C) at least of the active principles (biguanide ortransporter) is attached to the biogenic vectorization compound V usinga salification reaction, the sequence for carrying out the reactionswill preferably comprise the condensation reaction and then thesalification reaction, for reasons of stability of the salts as afunction of the pH, well known to those skilled in the art.

When A is metformin, C is arginine and V is succinic acid, thepreparation method comprises the following steps:

-   -   reaction of the monochloride monoester of the succinic acid in        solution in ether or in benzene, with the arginine in aqueous        solution in sodium carbonate,    -   release of the metformin base from the hydrochloride in        concentrated sodium hydroxide medium and extraction with        absolute alcohol,    -   formation of the salt of arginine hemisuccinimide with        metformin.

Preferably, the pharmaceutical compositions according to the inventionare adapted in a form which is suitable for oral, parenteral orintravenous administration.

A subject of the invention is more particularly the use of at least oneactive compound as described above, for producing medicines intended forthe treatment of diabetes in all its forms and/or for the treatment ofdiseases of the circulatory system, whether or not these diseases areattached to diabetes.

The present invention is now described byway of example, with referenceto the combination of metformin (biguanide) and arginine (transporter)in a same active compound A′V′C′, V being succinic acid reacting, on theone hand, covalently with an amine function of arginine and, on theother hand, ironically (salification [sic] reaction) with an aminefunction of metformin

Synthesis of Arginine Hemisuccinimide Metformin Hemisuccinate

-   -   a) First step: preparation of the arginine hemisuccinimide.

Arginine base (6 g) is dissolved in 120 ml of an aqueous solution ofsodium carbonate (N=10.6 g/100 ml). Moreover, succinic monochloridemonoester is diluted in 50 ml of sulfuric ether, with a slight excess ofsuccinic monochloride monoester for a reaction which is mole for molewith respect to the arginine. The ether-containing solution is added tothe aqueous solution in 10 minutes, with vigorous stirring at roomtemperature. The reaction liquid is maintained with vigorous stirringfor one hour, while slowly heating for complete distillation of theether. The mixture is evaporated to dryness, and the residue is taken upwith a minimum volume of distilled water (20 ml) and acidified withdiluted hydrochloric acid. By concentrating (slight heating underpartial vacuum) white crystals of arginine hemisuccinimide are obtained.

The NMR spectrum, the centesimal analysis and the purity of the productby thin-layer chromatography are verified. In particular, the presenceof the arginine amino acid residue is verified by the ninhydrin reactionand the presence of the free carboxyl of the succinic acid is verifiedby titrimetry.

The yield is quantitative.

b) Second step: release of the metformin base.

10 grams of metformin hydrochloride are added to 40 ml of a 5N sodiumhydroxide solution. The reaction mixture is heated for two hours at 40°C. After evaporation under vacuum at 40° C., the viscous residue istaken up with 100 ml of absolute ethanol. Filtration allows theimpurities to be eliminated and an insoluble residue of sodium chlorideremains. The metformin base is in alcoholic solution and it is isolated,by evaporation, in the form of a viscous powder. The NMR spectrumconfirms the structure of the metformin. The absence of chloride isverified with silver nitrate.

It is recalled that metformin, i.e. N,N-dimethylimidodicarbonimidicdiamide is identified in the MERCK Index under the number 5792 andcharacterized under the Chemical Abstracts number 657-24-9.

c) Third step

The metformin base is added, mole for mole, to an aqueous solution ofarginine hemisuccinimide. Immediate dissolving is obtained.

The water is completely evaporated off at 60° C. under vacuum. Theresidue is redissolved in distilled water and crystallizes duringconcentration under vacuum.

Translucent crystals which are soluble in water and insoluble in organicsolvents are obtained. The melting point is 188-189° C.

The NMR spectrum, the centesimal analysis and the presence of a singlespot after thin-layer chromatography confirm the structure and purity ofthe product. The total yield is quantitative.

After the above reactions, the yield is close to 90%. The lossesoriginate from the purifications and filtrations.

The developed formulae of the arginine, of the metformin and of the saltof the arginine hemisuccinimide with the metformin are given in FIGS. 1to 3, respectively.

Cleavage Assay:

This assay is carried out according to the in vitro method in anintestinal juice, described above, according to the in vitro toxicityassay described. Immediate release of the metformin without modifyingthe arginine hemisuccinimide part is observed. A second assay is carriedout on a rat hepatocyte culture, according to the method describedabove. A slow release of arginine over 24 hours is observed.

Toxicity:

This assay is carried out according to the in vitro toxicity assaydescribed above. The toxic dose is observed with the metformin at 10⁻²M, and it is identical for the active compound A′-V′-B′, namely the saltof the arginine hemisuccinimide with the metformin.

Verification of the Pharmacological Activity of the Active CompoundObtained

The kinetic and pharmacological advantage of the active compoundaccording to the present invention is described hereinafter, taking, byway of illustrative example, the arginine hemisuccinimide-metforminhemisuccinate, and a metformin hydrochloride/arginine hydrochloridecombination:

a) a pharmacokinetic study carried out in two groups, each of 20 rats,receiving, orally, 50 mg/kg of metformin hydrochloride and 50 mg/kg ofarginine hemisuccinimide-metformin hemisuccinate, respectively, made itpossible to calculate the various kinetic parameters. The argininehemisuccinimide-metformin hemisuccinate releases metformin and, in thetwo groups, it is the plasmatic levels of the metformin which aredetermined.

After administration of 50 mg/kg of metformin hydrochloride, theconcentration peak is observed within 90 minutes and is found to be 3.9μg/ml. The bioavailable fraction is 60% and the half-life is, onaverage, 2.5 hours.

The administration of 50 mg/kg of arginine hemisuccinimide-metforminhemisuccinate corresponds to approximately 25 mg/kg of metforminhydrochloride, i.e. to a half-dose. The concentration peak is observedat 60 minutes and it is found to be 2.9 μg/ml of metformin. Thebioavailable fraction is 75% and the half-life is 2.6 hours.

These results demonstrate that the entry of the metformin (total amountand rate of transfer) is improved in the case of the argininehemisuccinimide-metformin hemisuccinate.

From the pharmacological point of view, the anti-diabetic activity wasstudied on two models of rats made diabetic.

The first model consisted in treating the rate with streptozotocin (50mg/kg, IP), this being a compound which induces an increase in glycemia,which increases from 5.5 mM to 12-14 mM in 21 days. The administrationof metformin (30 mg/kg) significantly decreases this hyperglycemia,which decreases from 12.11 to 9.85 mM on average. At the same dose of 30mg/kg (approximately two times less metformin base), the argininehemisuccinimide-metformin hemisuccinate decreases more considerably thehyperglycemia, which decreases from 12.66 to 7.56 mM. The differencebetween the two treatments is significant despite the lower dose ofmetformin.

The second model is prepared by administering fructose at 10% in thedrinking water of the rats for three weeks. An insulin resistancedevelops, followed by diabetes of non-insulin-resistant type. Thearginine hemisuccinimide-metformin Hemisuccinate proves to besignificantly more active than the metformin alone, at an equivalentdose of metformin base and the action is more rapid as shown in Table 1below

TABLE 1 Arginine hemisuccinimide- metformin Metformin Argininehemisuccinate 30 mg/kg/day 30 mg/kg/day 70 mg/kg/day Control 6.94 ± 0.136.87 ± 0.14 6.84 ± 0.06 (glycemia mM) 21 days fructose 12.52 ± 0.52 13.24 ± 0.62  12.14 ± 0.28  (glycemia mM)  4 days treatment 13.41 ±0.52  14.37 ± 0.20  10.92 ± 0.44  (glycemia mM)  8 days treatment 10.83± 0.64  12.56 ± 0.89  9.13 ± 0.64 (glycemia mM) 12 days treatment 9.78 ±0.72 11.98 ± 0.71  8.43 ± 0.52 (glycemia mM) 21 days treatment 9.87 ±0.68 10.73 ± 1.27  7.76 ± 0.78 (glycemia mM)

The anti diabetic activity was also tested on hamsters made diabetic bythe administration of fructose for three months. On this model, thearginine hemisuccinimide-metformin hemisuccinate reveals itself to besignificantly more active than metformin on its own at an equal dose of10 mg/kg/day for both products after two weeks of treatment the resultsillustrated in the following Table 2 are obtained:

TABLE 2 Fructose + arginine Fructose + hemisuccinimide- Fructosemetformin metformin hemisuccinate 3 months 10 mg/kg/day 10 mg/kg/day 2weeks treatment 143 112 93 (glycemia mg/dl) Control glycemia withoutfructose: 91 mg/dl

A study on the cheek pouch of the hamster shows that the argininehemisuccinimide-metformin hemisuccinate reproduces at least the effectsof the two active principles on the microcirculation, namely thevasodilatory action of the arginine and the action of the metformin onvasomotion.

1. A compound of formula III


2. A method for treating diabetes in mammals, comprising administering acompound of formula III as claimed in claim
 1. 3. A process forpreparing an active compound of formula III as claimed in claim 1,comprising the following steps: (a) reaction by condensation and/orsalification of succinic acid with one of arginine and metformin, and(b) reaction by condensation and/or salification of the product of thereaction according to (a) with the other of arginine and metformin.